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M L Gubler

Publications and source records attributed to M L Gubler.

11 recordsLinked to original sources
PubMed1996

Restoration of the transcription activation function to mutant p53 in human cancer cells.

The p53 tumor suppressor gene product is a sequence-specific transcription activator frequently mutated in a variety of human malignancies. Typically, tumor-derived p53 missense mutants are defective in DNA binding and this is likely to result in a failure to active p53-regulated genes. Hence, restoring function to mutant p53 represents an attractive target to develop a novel cancer chemotherapeutic agent. We now show that a small chemically modified peptide derived from p53 restores sequence-specific DNA binding to a subset of p53 mutants. Moreover, when microinjected into human colon carcinoma cells this peptide restores the transcription activation function to endogenous mutant p53 protein. This is the first example showing that a small peptide molecule can reverse the effect of several inactivating missense mutations and restore protein function.

Amino Acid Sequence↗
PubMed1995

Microinjection of monoclonal antibody PAb421 into human SW480 colorectal carcinoma cells restores the transcription activation function to mutant p53.

The p53 tumor suppressor is a transcription factor frequently mutated in human malignancies. Tumor-derived p53 missense mutants are defective in sequence-specific DNA binding and fail to activate p53 target genes. mAb PAb421 was shown previously to restore DNA binding to selected p53 mutants in vitro. Here we show that mAb PAb421 when microinjected into human SW480 colorectal carcinoma cells restores the transcription activation function to the resident mutant p53 (arg to his 273, pro to ser 309). Codon 273 is the second most frequent p53 missense mutant found in human tumors. Our results lend support to the concept of restoring wild-type function to mutant p53 as a strategy for cancer therapy.

Antibodies, Monoclonal↗
PubMed1995

Nonradioactive assay for sequence-specific DNA binding proteins.

A nonradioactive functional assay was developed to quantitate DNA binding proteins. The assay was designed to allow the use of 96-well microplates for high sample throughput. We show that the assay can measure sequence-specific DNA binding of purified proteins as well as DNA binding activity present in whole cell extracts. By using a temperature-sensitive DNA binding protein, we demonstrate that DNA binding as measured by this novel assay correlates with the biological function of the protein.

Animals↗
PubMed1988

Effects of dietary retinoids upon growth and differentiation of tumors derived from several murine embryonal carcinoma cell lines.

We have examined the effects of dietary retinoids upon the growth and differentiation of seven embryonal carcinoma lines in mice. The control diet contained 4000 IU/mg retinyl palmitate; the other diets contained 2 x 10(5) IU/mg retinyl palmitate, 50 mg/kg all-trans-retinoic acid (RA), 100 mg/kg RA, and no retinoid. The RA-containing diets had little influence on tumor latency or incidence but did suppress growth of many of the tumors. Decreased tumor mass was, in most but not all instances, accompanied by an increased proportion of differentiated cells. Increased differentiation was most commonly quantitative rather than qualitative; i.e., there was a larger proportion of the same types of differentiated cells seen in tumors from the control diet group rather than an increase in the spectrum of cell types observed. Notably, tumors from two differentiation-defective embryonal carcinoma lines were refractory to both the differentiation-inducing and growth-suppressing properties of dietary RA. Taken together, our results suggest that dietary RA can reduce teratocarcinoma growth in part by promoting differentiation but that other mechanisms are likely to be involved. The therapeutic benefits that we observed with dietary RA were compromised by adverse effects, including failure of the mice to gain weight as effectively as those on the control diet. The effects of elevated levels of retinyl palmitate, or its omission from the diet, were much less striking than that of RA. Both modifications tended to decrease tumor latency but had little effect, if any, upon ultimate tumor mass. Elimination of retinoid from the diet failed to significantly reduce degree of differentiation in tumors which normally differentiate extensively in animals on retinoid-containing diets. Excess retinyl palmitate led to a marginal increase in differentiation in F9 tumors and a statistically significant increase in differentiation in OC15-S1 tumors. Tumors from other embryonal carcinoma lines did not contain elevated levels of differentiated cells. The interpretation of these results is complicated by our observations that although our dietary alteration in levels of palmitate were dramatic, they resulted in much more modest differences in circulating retinoid levels when compared with mice on the control diet.

Animals↗
PubMed1987

Differential uptake, binding, and metabolism of retinol and retinoic acid by 10T1/2 cells.

Previous studies have shown that all-trans-retinoic acid fails to inhibit chemically induced transformation in 10T1/2 cells except at toxic levels, whereas retinol and many synthetic retinoids are potent inhibitors. In contrast, in many systems retinoic acid is a more effective modulator of differentiation and carcinogenesis than is retinol. In any attempt to explain this anomaly, we have studied the differential metabolism of retinoic acid and retinol by 10T1/2 cells and by their initiated and transformed derivatives, and have also reexamined these cells for the presence of retinoid-binding proteins. Whereas retinoic acid was depleted from the medium bathing 10T1/2 and initiated 10T1/2 cells within 48 h of treatment, retinol was concentrated 500-fold by these cells, and disappeared from the culture medium no faster than from cell-free medium. Retinoic acid metabolism by a number of transformed cell lines varied widely. There was no apparent correlation between metabolizing ability and transforming agent (methylcholanthrene, X-rays, fission spectrum neutrons, and plasmid oncogene transfection). Uptake of retinoic acid was seen in all cell lines and was not correlated with its metabolism. Retinol was metabolized minimally by all cell lines tested; metabolism of retinol was not correlated with retinoic acid metabolizing ability. Retinoic acid-induced growth inhibition and cytotoxicity were not correlated with metabolizing ability, suggesting that the rate of metabolism of retinoic acid is not a major determinant of its acute biological effects. Using sensitive radioimmunoassays, cellular retinoic acid- (CRABP) and retino-binding proteins (CRBP) were both detected in 10T1/2 and initiated 10T1/2 cells. CRABP levels of about 16 ng/10(6) cells were about 4-fold higher than CRBP levels. Therefore, lack of CRABP does not explain the failure of retinoic acid to inhibit carcinogen-induced transformation in these cells. These studies suggest that the inability of retinoic acid to inhibit transformation in the 10T1/2 cell system may be due to its rapid metabolism and clearance from the medium. On the other hand, the high cellular uptake and stability of retinol in these cells could be an important factor in the inhibition of neoplastic transformation by this retinoid.

Carrier Proteins↗
PubMed1985

Metabolism of retinoids by embryonal carcinoma cells.

Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.

Animals↗
PubMed1985

Role of retinoids in differentiation and growth of embryonal carcinoma cells.

To study how retinoids promote differentiation and inhibit proliferation of embryonal carcinoma (EC) cells, we have followed their intracellular fate. Retinoic acid (RA) is effectively metabolized to more polar compounds by many EC lines. Unlike RA, retinol is slowly metabolized. Our inability to detect conversion of retinol to RA might indicate that the two retinoids elicit their effects on EC cells in different ways. Retinol added to cultures quickly appears in the nuclear fraction; the proportion associated with nuclei after detergent extraction is initially very low but increases with time. Retinol and RA might be translocated to nuclei by their respective binding proteins [cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP)]: isolated EC nuclei have specific, independent binding sites for both holoproteins but not their ligands. CRABP cannot be detected in the nucleoplasm of untreated EC cells, but activity is measurable after cells are exposed to RA. Interestingly, incubation with retinol promotes movement of both CRBP and CRABP into the nucleoplasmic fraction. Finally, we have demonstrated that brief exposure to RA dramatically reduces the cloning efficiency of EC cells. Since some cells are unaffected even by lengthy exposures to RA whereas the growth of their progeny is inhibited, we suggest that EC cells can become epigenetically refractory to RA.

Animals↗
PubMed1984

Complementation analyses of differentiation-defective embryonal carcinoma cells.

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.

Acetamides↗
PubMed1984

Sodium butyrate induces histone hyperacetylation and differentiation of murine embryonal carcinoma cells.

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.

Acetylation↗